The project focuses on molecular mechanisms by which enzymes oxdize hydrogen gas and reduce protons to make hydrogen. The processing of hydrogen is employed by many micro-organisms including pathogenic ones and involves extremely unusual cofactors and active sites. The work provides mechanistic insights into the newly realized biological function of iron, i.e. where the metal operates at lower oxidation states and is supported by unusual ligands such as CO and cyanide. Mechanistic characterization of these replicas of the active site of the Fe-only hydrogenase will provide insights unavailable by classical enzymological characterization. Preliminary studies show that oxidative decarbonylation of diiron dithiolato carbonyls affords species that bear a close structural resemblance to the enzyme's active sites, such as the previously unobserved bridging CO ligand. The project will build on these initial successeswith the goal of generating species with hydrogenic substrates bound as predicted by mechanistic enzymologists. We will probe for the first examples of mixed valency in reduced Fe-S systems. The new diferrous models will be employed to generate the first iron dihydrogen species, directly relevant to the catalytic mechanism. Redox auxiliaries will be attached to the diiron center to probe the kinetic and thermodynamic benefits of coupling the electron donor and the proton receptor. The mechanistic role of the associated cofactors - both the redox reservoir Fe-S protein and azadithiolate - will be probed. The newly developed methodologies will be applied to related mechanistic and preparative challenges related to the NiFe hydrogenases: the biosynthesis of the cyanide cofactor, metallocenethiolates to confer novel geometries, and second coordination sphere control of proton availabilit.